Processing of acylchitosan hydrogels

ABSTRACT

An article containing N-acylchitosan is manufactured by a process comprising the steps of providing a mixture containing chitosan and/or N-acylchitosan, and extruding the mixture to form an N-acylchitosan hydrogel. Alternatively, the process comprising the steps of providing a chitosan and/or N-acylchitosan hydrogel, and extruding the hydrogel. An article with a memorized shape is formed by fixing the N-acylchitosan hydrogel in a desired shape, and at least partially drying the fixed hydrogel. A patient is treated by injecting the N-acylchitosan hydrogel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. national stageapplication Ser. No. 12/067,727 filed May 28, 2008, which is a NationalStage application of PCT/EP2006/009830, filed Oct. 11, 2006, whichclaims priority to U.S. Provisional Application Nos. 60/725,575, filedOct. 12, 2005 and 60/725,610, filed Oct. 12, 2005. The entire contentsof each of the aforementioned applications are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to a method of extruding products based onN-acylchitosan, and articles made thereof, including shaped medicaldevices having improved mechanical strength and shape-memory stability.

BACKGROUND OF THE INVENTION

Chitin and chitosan represent a family of biopolymers, made up ofN-acetyl-D-glucosamine and D-glucosamine subunits. Chitin can be foundwidely in the exoskeletons of arthropods, shells of crustaceans, and thecuticles of insects. Chitosan, although occurring in some fungi, isproduced industrially by alkaline hydrolysis of chitin. Their differentsolubilities in dilute acids are commonly used to distinguish betweenboth polysaccharides. Chitosan, the soluble form, can have a degree ofacetylation between 0% and about 60%, the upper limit depending onparameters such as processing conditions, molar mass, and solventcharacteristics.

Both chitin and chitosan are promising polymers for a variety ofapplications, including water treatment (metal removal,flocculant/coagulant, filtration), pulp and paper (surface treatment,photographic paper, copy paper), cosmetics (make-up powder, nail polish,moisturizers, fixtures, bath lotion, face, hand and body creams,toothpaste, foam enhancing), biotechnology (enzyme immobilization,protein separation, chromatography, cell recovery, cell immobilization,glucose electrode), agriculture (seed coating, leaf coating,hydroponic/fertilizer, controlled agrochemical release), food (removalof dyes, solids and acids, preservatives, color stabilization, animalfeed additive), and membranes (reverse osmosis, permeability control,solvent separation). Of particular interest are biomedical applicationsof chitin and chitosan because of their biocompatibility,biodegradability and structural similarity to the glycosaminoglycans.Applications and potential applications include dressings forwound-healing, tissue engineering applications, artificial kidneymembranes, drug delivery systems, absorbable sutures, hemostats,antimicrobial applications, as well as applications in dentistry,orthopedics, ophthalmology, and plastic surgery. For comprehensivereviews of potential applications of chitin and chitosan see, forexample, [Applications of chitin and chitosan, 1997. Shigemasa andMinami, Biotech Genetic Eng Rev 1996; 13:383-420. Ravi Kumar, ReactFunct Polym 2000; 46:1-27. Singh and Ray, J Macromol Sci 2000;C40:69-83].

However, despite a great variety of potential applications of chitin andchitosan, only a few products are actually in commercial use. One of themajor limiting factors for a still broader utilization is the limitedcapability for extruding these polysaccharides in an efficient manner toproducts having the desired properties. As an example, chitin andchitosan fibers are usually made by wet-spinning processes, whichproduce fibers by first dissolving the polymer in a solvent and thenextruding the polymer solution into a nonsolvent. However, chitin isinsoluble in common solvents, which prevents facile processing. Forexample, surgical suture made of chitin fiber has been described in U.S.Pat. No. 3,988,411 to Capozza and U.S. Pat. No. 4,932,404 to Kifune etal. which is fabricated by wet-spinning processes using toxic,corrosive, and expensive halogenated solvents. N,N-dimethylacetamidecontaining lithium chloride has been shown to be an effectivealternative solvent system for chitin, overcoming some of the issuesassociated with halogenated solvents. For example, as described in U.S.Pat. No. 4,059,457 to Austin, chitin fibers can be fabricated using thissolvent system by extrusion into an acetone coagulation bath. However, ageneral problem remains with the removal of the lithium chloride fromthe fiber. The lithium acts as a Lewis acid solvating the chitin amidegroup, and it is unclear if this can be completely reversed throughwashing, once the fiber has been formed. These issues as well as generalaspects of chitin fiber processing and solvent systems have beenreviewed thoroughly [Rathke and Hudson, J Mater Sci 1994; C34:375437.Agboh and Qin, Polym Adv Tech 1997; 8:355-365. Ravi Kumar, React FunctPolym 2000; 46:1-27].

Chitosan is more readily soluble, and fibers can be prepared byextrusion of diluted acidic solutions of chitosan into an alkalinecoagulation bath, such as described in U.S. Pat. No. 2,040,880 to Rigby.However, chitosan fibers fabricated in this manner have low mechanicalstrength in physiological environment, requiring a subsequent covalentcross-linking procedure [Knaul et al., J Polym Sci 1999; B37:1079-1094].Methods to improve the mechanical strength of chitosan articles, such asfibers and tubes, have also been suggested in U.S. Pat. No. 6,368,356 toZhong et al., by using a combined ionic and covalent cross-linkingprocess. However, covalent cross-linking regularly involves toxicchemical substances and by-products which may be difficult to removefrom the product. Cross-linking also alters the natural chemicalstructure of the biopolymer, thereby affecting natural biodegradationprocesses and products. Additionally, the mechanical strength ofionically and/or covalently cross-linked chitosan is still poor underphysiological conditions, and articles having a memorized shape such asthose described in '356 quickly loose their shape under physiologicalconditions.

In “Study of a chitin-based gel as injectable material in periodontalsurgery”, Biomaterials 2002; 23:1295-1302, Gerentes et al. disclose atreatment of periodontal disease by means of injecting a mixturecontaining chitosan and acetic anhydride before gelation. In“Chitin-based tubes for tissue engineering in the nervous system”,Biomaterials 2005; 26:4624-4632, Freier et al. disclose a method ofmanufacturing tubes by means of injecting a mixture containing chitosanand acetic anhydride into a mold before gelation.

By considering the aforementioned limitations in the prior art it wouldbe advantageous to manufacture chitin/chitosan-based fibers, tubes andother articles by a simple, inexpensive and efficient process, withoutthe use of toxic solvents and cross-linking agents, and without therelease of toxic by-products. It would further be advantageous tomanufacture chitin/chitosan-based fibers, tubes and other articles by anextrusion process, leading to sufficient mechanical strength of theextruded products under physiological conditions. It would further beadvantageous to manufacture chitin/chitosan-based shaped articles whichhave an improved mechanical stability under physiological conditions,including a mechanically stable shape-memory which allows the article tobe reversibly shaped in different conformations. It would further beadvantageous to manufacture chitin/chitosan-based shaped articles whichallow for controlled degradation and/or dissolution to non-toxicproducts under physiological conditions. These and other needs are metin the present invention.

SUMMARY OF THE INVENTION

In the description of the present invention, the term “chitin” is usedfor a naturally derived polymer made up of either N-acetyl-D-glucosaminesubunits or N-acetyl-D-glucosamine and D-glucosamine subunits that isnon-soluble in dilute acids. The term “chitosan” is used for a polymermade up of either D-glucosamine subunits or N-acetyl-D-glucosamine andD-glucosamine subunits that is either naturally derived or syntheticallyprepared by hydrolysis of chitin and that is soluble in dilute acids.The term “N-acylchitosan” represents a polymer that is syntheticallyprepared by N-acylation of chitosan or that is synthetically prepared byN-acylation or hydrolysis of an N-acylchitosan prepared by N-acylationof chitosan. The term “N-acylchitosan hydrogel” is used for anN-acylchitosan network that is swollen in an aqueous environment. Theterm “acylation” is used for the N-acylation of the amine group ofchitin, chitosan or N-acylchitosan. Accordingly, “acetylation” describesthe N-acetylation of chitin, chitosan or N-acylchitosan. The term“deacylation” is used for the N-deacylation of the amide group ofchitin, chitosan or N-acylchitosan. Accordingly, “deacetylation”describes the N-deacetylation of chitin, chitosan or N-acylchitosan.

It is an object of the present invention to provide a better process ofmanufacturing an article containing N-acylchitosan.

It is a further object of the present invention to provide an extrusionprocess to manufacture chitin/chitosan-based products which overcomeslimitations in the prior art, such as the use of toxic solvents andcross-linking agents, as well as insufficient mechanical strength ofextruded products under physiological conditions.

In accordance with the present invention, there are provided processesof manufacturing an article containing N-acylchitosan: a process ofmanufacturing an article containing N-acylchitosan, the processcomprising the steps of providing a mixture containing chitosan and/orN-acylchitosan, and extruding the mixture to form an N-acylchitosanhydrogel; a process of manufacturing an article containingN-acylchitosan, the process comprising the steps of providing a chitosanand/or N-acylchitosan hydrogel, and extruding the hydrogel; and aprocess of manufacturing an article containing N-acylchitosan, theprocess comprising the steps of providing an N-acylchitosan hydrogelfixed in a desired shape, and at least partially drying the hydrogel,thereby forming an article with a memorized shape.

In accordance with the present invention, there is provided an extrusionprocess which may comprise of: a) providing a mixture containingchitosan and/or N-acylchitosan, and b) extruding the mixture, therebyessentially instantly forming an N-acylchitosan hydrogel.

In accordance with the present invention, there is provided an extrusionprocess which may comprise the extrusion of the mixture into ahydrogelation medium capable of promoting the formation of anN-acylchitosan hydrogel.

In accordance with the present invention, there is provided an extrusionprocess which may comprise the extrusion of a mixture containingchitosan into a mixture containing an acylation agent, therebyessentially instantly forming an N-acylchitosan hydrogel upon extrusion.

In accordance with the present invention, there is provided an extrusionprocess which may comprise the addition of an acylation agent to thechitosan mixture prior to extrusion in order to increase the degree ofacylation of chitosan thereby facilitating the gel formation uponextrusion.

In accordance with the present invention, there is provided an extrusionprocess which may involve a mixture, solution, or gel extruded.

In accordance with the present invention there is provided an extrusionprocess which may comprise the steps of providing a chitosan and/orN-acylchitosan hydrogel having shear-thinning properties and extrudingthe hydrogel.

In accordance with the present invention there is provided an extrusionprocess which may comprise the steps of providing a chitosan and/orN-acylchitosan hydrogel and extruding the hydrogel, and wherein prior toextrusion the components of the chitosan and/or N-acylchitosan hydrogelmay have essentially completely reacted.

In accordance with the present invention, there is provided an extrusionprocess which may involve hydrolysis of the extruded product, therebyforming a product having improved mechanical strength.

In accordance with the present invention, there is provided an extrusionprocess which may involve at least partial drying of the extrudedproduct, thereby forming a product having improved mechanical strength.

In accordance with the present invention, there is provided an extrusionprocess which may involve coating of the extruded product, therebyforming a product having improved mechanical strength.

In accordance with the present invention, there is provided an extrusionprocess which may involve one or more of the aforementionedmodifications of the extruded product, thereby controlling itsmechanical, biocompatibility and biodegradation properties.

In accordance with the present invention, there is provided an extrusionprocess which may be suitable for the fabrication of N-acylchitosangels, fibers, tubes, films, and other articles.

It is further an object of the present invention to provide a shapedarticle based on N-acylchitosan which overcomes limitations in the priorart, such as the use of toxic substances and release of toxicby-products during processing, poor stability of mechanical propertiesand shape memory, and limited control of degradation and dissolutionwhen used as a temporary implant material.

It is another object of the present invention to provide a bettermethod, product and system for the medical treatment of a patient.

In accordance with the present invention, there is provided a shapearticle. Further in accordance with the present invention, there areprovided systems for the medical treatment of a patient: a system forthe medical treatment of a patient, characterized in that the systemcomprises an injectable N-acylchitosan hydrogel, and means for injectingthe N-acylchitosan hydrogel into the patient; and a system fordelivering a therapeutic agent into a patient, characterized in that thesystem comprises an injectable N-acylchitosan hydrogel comprising thetherapeutic agent, and means for injecting the N-acylchitosan hydrogelinto the patient. Further in accordance with the present invention,there are provided methods for the medical treatment of a patient; amethod of treating a patient comprising the steps of providing anN-acylchitosan hydrogel, and injecting the N-acylchitosan hydrogel intothe patient; and a method of delivering a therapeutic agent comprisingthe steps of providing an N-acylchitosan hydrogel comprising thetherapeutic agent, and injecting the N-acylchitosan hydrogel into apatient. Further in accordance with the present invention, there areprovided uses of an injectable N-acylchitosan hydrogel; use of aninjectable N-acylchitosan hydrogel for the medical treatment of apatient; and use of an injectable N-acylchitosan hydrogel comprising atherapeutic agent for delivering the therapeutic agent into a patient.Finally in accordance with the present invention, there is provided aninjectable N-acylchitosan hydrogel comprising a therapeutic agent.

In accordance with the present invention, there is provided a shapedarticle, which is made by providing an N-acylchitosan gel, which isfixed in the desired shape, and which is dried under controlledconditions to memorize the shape.

In accordance with the present invention, there is provided a shapedarticle made of N-acylchitosan, which may have a shape memory, allowingthe article to be reversibly shaped in different conformations for easeof use such as implantation into a body.

In accordance with the present invention, there is provided a shapedarticle which may be made by at least partial drying of N-acylchitosangel having one or more of the following structures: rod, fiber, tube,film, sphere or other geometric structures which may be hollow or not.

In accordance with the present invention, there is provided a shapedarticle which may be made by at least partial drying of N-acylchitosangel, which is conformable to the shape of a medical device or part of amedical device, including the shape of an anchor, hook, coil, mesh,textile, foam, scaffold, stent, catheter, tube, sphere, particle, plug,plate, screw, pin, tack, clip, ring, drug-release depot,cell-encapsulation device.

In accordance with the present invention, there is provided a shapedarticle which may be a coating, made by at least partial drying ofN-acylchitosan gel, of a medical device or part of a medical device,including an anchor, hook, fiber, coil, mesh, textile, foam, sponge,scaffold, stent, catheter, tube, sphere, particle, plug, plate, screw,pin, tack, clip, ring, drug-release depot, cell-encapsulation device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one preferred design of the extrusion processincluding optional steps of hydrolysis, drying, and coating.

FIG. 2 is an illustrative example of a selective N-acylation reactionforming N-acetylchitosan hydrogels. Gelation occurs by addition of morethan approximately 0.7-fold acetic anhydride to the chitosan solution(per glucosamine unit of chitosan, degree of acetylation at start 11%),which is equivalent to a degree of acetylation greater thanapproximately 70% (shadowed area).

FIG. 3 illustrates the hydrolysis of N-acetylchitosan tubes with 40%aqueous sodium hydroxide solution at 110 .degree. C.

FIG. 4 illustrates typical load-displacement curves of N-acetylchitosantubes (in phosphate buffered solution, 37.degree. C.) before and afterone, two, or three hydrolysis cycles (for 2 h each) with 40% aqueoussodium hydroxide solution at 110.degree. C.

FIG. 5 illustrates typical load-displacement curves of N-acetylchitosangel and air-dried tubes (in phosphate buffered solution, 37.degree. C.).

FIG. 6 is an illustration of a shaped medical device made fromN-acylchitosan fiber according to the present invention.

FIGS. 7 and 8 each is an illustration of another shaped medical devicemade from N-acylchitosan tube according to the present invention.

FIGS. 9A and 9B each is an illustration of a shaped medical device madefrom a combination of a two-channel tube and a hollow sphere accordingto the present invention. FIG. 9A is a side-view of a part of thedevice, and FIG. 9B is a cross-section of the tube.

FIG. 10 is an image (side-view) of a coil-shaped medical device which iscoated with N-acylchitosan according to the present invention.

FIGS. 11 and 12 each is an image (cross-section) of a cylindricalmedical device which is coated with N-acylchitosan according to thepresent invention. FIG. 11 illustrates a coated solid, and FIG. 12 acoated hollow, part of a device.

FIG. 13 is an image (side view) of a screw-shaped medical device whichis coated with N-acylchitosan according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

In one preferred embodiment, the invention relates to a method ofextruding articles based on N-acylchitosan. More particularly, theinvention relates to an extrusion process for the fabrication of gels,fibers, tubes, films, and other articles. Extruded articles according tothe present invention may include non-medical and medical products.Additionally, extruded articles according to the present invention maybe used to fabricate non-medical and medical products. Non-medicalproducts which may consist in total or in part of an extruded articleaccording to the present invention may include separation membranes (gasseparation, dialysis, reverse osmosis, ultrafiltration), affinityfiltration membranes (chromatography), encapsulation membranes (cells,enzymes), coatings in agriculture (seeds, agrochemicals), water and airtreatment products (removal of heavy metals and dyes, air-cleaners),textiles, cosmetics, sanitary products (diapers, panty liners, tampons),food additives, and others. Medical products which may consist in totalor in part of an extruded product according to the present invention mayinclude sutures, suture fasteners, slings, coils, rivets, tacks,staples, clips, hooks, buttons, snaps, orthopedicpins/clamps/screws/dowels/plates, bone substitutes, spinalcages/plates/rods/screws/discs, finger joints, intramedullary nails, hipprosthesis, meniscus repair devices, knee replacement devices, cartilagerepair devices, ligament and tendon grafts, tendon repair devices,surgical mesh, surgical repair patches, hernia patches, pericardialpatches, cardiovascular patches, adhesion barriers, abdominal wallprosthesis, catheters, shunts, stents (coronary, gastrointestinal,esophageal, biliary, ureteral, urethral, stents for aortic aneurysms),vascular grafts and substitutes, coronary artery bypass grafts, guidedtissue repair/regeneration devices, scaffolds for tissue engineering,nerve guides, septal defect repair devices, heart valves, vein valves,artificial fallopian tubes, drainage tubes and implants, intrauterinedevices, intraocular implants, keratoprosthesis, dental implants,orbital floor substitutes, skin substitutes, dural substitutes,intestinal substitutes, fascial substitutes, wound dressings, burndressings, medicated dressings, gauze/fabric/sheet/felt/sponge forhemostasis, gauze bandages, bandages for skin surfaces, adhesivebandages, bulking and filling agents, drug delivery matrices, injectablegels and systems, coatings applied to pacemaker leads, implantablesensor wire leads, wires for interventional cardiology, or biosensors,and others.

The extruded articles according to the present invention are made byN-acylation of chitosan forming N-acylchitosan gels. The selectiveN-acylation reaction of chitosan forming N-acylchitosan gels iswell-known in the art and usually includes the treatment of chitosan,which is dissolved in diluted acidic solution and mixed with acosolvent, with an acyl anhydride. After mixing of the components, gelformation occurs within a few seconds to hours, depending on thereaction conditions and used reactants. However, this method of gelformation known in the prior art is impracticable for application to anextrusion process which requires instant and continuous formation ofself-supporting structures under steady-state conditions. Mixture of allcomponents of the acylation reaction prior to the extrusion would beassociated with an inhomogeneous process characterized by changingproperties of the extruded mixture including increase in the solutionviscosity, beginning of gelation including formation of microgels in thesolution, and finally complete gelation, which is impracticable tocontrol in a commercial extrusion setup.

According to one preferred embodiment of the present invention, there isprovided an extrusion process comprising dissolution of chitosan in adiluted acid, addition of a cosolvent to the chitosan solution, andextrusion of the mixture into a solution containing an appropriateamount of an acylation agent, thereby essentially instantly forming anN-acylchitosan hydrogel upon extrusion. This inventive method allows foran extrusion under steady-state conditions as required for processessuch as wet-spinning of fibers and hollow fibers (tubes).

In a typical setup, the extrusion line is comprised of a reservoircontaining the chitosan solution which will be filtered (by passingthrough a filter either before adding to the reservoir or while leavingthe reservoir, as it is known in the art) and degassed (e.g. byevacuation or sonication, as it is known in the art) and which will beforced, by means of a pump, through a die (e.g. a single- or multi-holefiber spinneret, or a coaxial tube spinneret) into the acylation bathfor gelation (FIG. 1). Take-up rollers at the end of the acylation bathwill be maintained at a velocity which prevents kinking of theN-acylchitosan gel article formed. Following gelation in the acylationbath, the extruded article will be washed by passing through a bathcontaining water which is maintained at room temperature. The extrudedarticle may simultaneously (to the washing step) be subjected todrawing, by rotating the take-up roller at the end of the water bath ata higher speed than the advancing roller at the beginning of the waterbath. Drawing may be performed at room temperature or at elevatedtemperature. The extruded article may then be dried by passing through adrying bath containing one or more organic drying agents known in theart, such as acetone, ethanol and isopropanol, and/or air-dried byconvection at room temperature or elevated temperature, before beingwound up on a winder. Alternatively, as it will be outlined in detailfurther below, the extruded article may be subjected to hydrolysisand/or coating procedures before or after drying. Furthermore, as itwill be outlined in detail further below, drying may be performed insuch a way that a specific shape-memorized design will be implemented inthe extruded article.

The most preferable acylation agent for use according to the presentinvention is acetic anhydride; however, other acylation agents,including, for example, propionic, butyric, hexanoic, octanoic,decanoic, maleic, and methacrylic anhydride may be used for gelformation. Suitable solvents for chitosan include dilute inorganic andorganic acids, such as formic, acetic, propionic, lactic, and citricacid; most preferable to manufacture gels for use according to thepresent invention is aqueous acetic acid. Suitable cosolvents to beadded to the chitosan solution as well as solvents to be added to theacylation bath (i.e., to dissolve/dilute the acylation agent) includewater as well as organic liquids, such as methanol, ethanol, propanol,butanol, trifluoroethanol, ethylene glycol, diethylene glycol,polyethylene glycol, glycerol, formamide, N,N-dimethyl formamide,N-methylpyrrolidone, dimethyl sulfoxide, dioxane, and tetrahydrofurane.Most preferable to manufacture gels for use according to the presentinvention are ethanol and methanol. The well-known fact thatN-acylchitosan hydrogels are practically insoluble in most commonsolvents allows for gel formation in a broad variety of acylation bathsmade of different acylation agent/solvent mixtures, and the use ofmixtures of different acylation agents as well as solvent mixtures.

Preferred products extruded according to the present invention are thosebased on N-acetylchitosan. Preferably, the extrusion involvesdissolution of 2-10% chitosan in 0.5-15% aqueous acetic acid, additionof a 1-2.5 fold volume of ethanol, and extrusion of the resultinghomogeneous mixture into an acylation bath containing a solution of10-90% acetic anhydride in ethanol. More preferably, chitosan isdissolved in a concentration of 3-5% in 2-5% aqueous acetic acid, mixedwith a 1-2 fold volume of ethanol, and extruded into an acylation bathcontaining 25-50% of acetic anhydride in ethanol (Example 1).

Preferably, the chitosan used as starting material for extrusionprocesses according to the present invention has a degree of acetylationof less than 25% and a viscosity between approximately 200-2000 mPas(analyzed as 1% solution in 1% acetic acid on a Brookfield viscometer at25.degree. C.). More preferably, the chitosan has a degree ofacetylation of less than 15% and a viscosity between approximately200-1000 mPas.

Methanol may be beneficial over ethanol under specific conditions,particularly if higher concentrations of chitosan are used. In thiscase, chitosan is preferably dissolved in a concentration of 5-7% in2-5% aqueous acetic acid, mixed with a 1-2 fold volume of methanol, andextruded into an acylation bath containing 25-50% of acetic anhydride ineither ethanol or methanol (see Example 1). Extrusion may also beperformed into a pure acylation agent without addition of any solvent tothe acylation bath. The extrusion process may preferably be performed atroom temperature; however, higher temperatures, up to approximately60.degree. C., of the extruded mixture and/or acylation bath may be usedto accelerate the gelation process.

Another method to accelerate the gelation of the chitosan mixture uponextrusion into the acylation bath is to use chitosan having a highdegree of acylation of up to approximately 60%, or to incorporate apre-acylation step of chitosan to increase its degree of acylation priorto extrusion, by addition of an acylation agent to the chitosansolution/cosolvent mixture (see Example 1). The acylation agent used forpre-acylation may be equal or different to that used for the acylationbath. It is a preferred embodiment of the present invention that, inorder to allow for a homogeneous extrusion process, the components ofthe pre-acylation reaction have essentially completely reacted prior toextrusion. Pre-acylation is preferably performed by using an appropriateamount of acylation agent, so that the formed N-acylchitosan remainscompletely dissolved without gel formation prior to extrusion. Forexample, N-acetylchitosan gels are usually formed by addition of morethan 0.7 fold acetic anhydride to a solution of chitosan, having adegree of acetylation of 11% at start, in 2% aqueous acetic acid mixedwith an equal volume of ethanol, which is equivalent to a degree ofacetylation of the product greater than 70% (FIG. 2). According to thisexample, the amount of acetic anhydride for pre-acetylation should beless than 0.7 fold so that the formed N-acetylchitosan is less than 70%acetylated, thereby remaining soluble prior to extrusion. Moregenerally, pre-acetylation is preferably performed by addition of a0.3-0.7 fold molar amount of acetic anhydride to the chitosan solution(per glucosamine unit of chitosan). The amount of acyl anhydride otherthan acetic anhydride that may be added to a chitosan solution for thepurpose of pre-acylation without gel formation prior to extrusiondepends on the chemical nature. Generally, the more hydrophobic the acylanhydride the less the amount that may be added. The amount of acylanhydride that may be added to a chitosan solution for the purpose ofpre-acylation without gel formation prior to extrusion also depends onthe amount of cosolvent added to the chitosan solution. Generally, thehigher the cosolvent content the less the amount of acyl anhydride thatmay be added.

Alternatively, pre-acylation may result in a gel which is extrudableutilizing the shear-thinning properties of N-acylchitosan gels. In thislatter case, extrusion into an acylation bath may be applied if productsof high mechanical strength are desired. However, in specificapplications, extrudates having low to moderate mechanical strength maybe sufficient so that extrudable gels may be applied without the step ofextrusion into an acylation bath. Such an example may be an injectablesystem for application in a patient for the purpose of filling orbulging, or to deliver therapeutic agents, taking advantage of theshear-thinning properties of N-acylchitosan gels, which enable thesegels to be extrudable directly through a needle via a syringe-extrusionin a steady-state manner. N-acylchitosan gels having appropriateshear-thinning properties making them suitable for injection are thosehaving moderate degrees of acylation, usually below 90% and preferablybelow 80%, in order to decrease injection forces. Among N-acylchitosangels, N-acetylchitosan gels are preferably used as injectable gelsaccording to the present invention. For example, an N-acetylchitosan gelwhich is formed by addition of a 0.7 fold molar amount of aceticanhydride to a 3% chitosan solution in 2% aqueous acetic acid mixed withan equal volume of ethanol can easily be applied using a 30 gaugeneedle. The amount of acetic anhydride required for gel formationdepends on the amount of cosolvent added, as outlined above. Thus forexample, addition of a 0.5 fold amount of acetic anhydride to a solutionof 3% chitosan in 2% aqueous acetic acid mixed with a twofold amount ofethanol likewise results in an N-acetylchitosan gel extrudable through a30 gauge needle (Example 2).

In a preferred embodiment of the present invention, prior to extrusionthe components of the chitosan and/or N-acylchitosan hydrogel haveessentially completely reacted. In this context, “completely reacted”refers to the acylation of the 2-amino-group of the chitosan and/orN-acylchitosan. It can be an advantage of this embodiment of theinvention that the hydrogel is of a well-defined consistency regardlessof the exact time of extrusion.

Applications of injectable gels made of N-acylchitosan may be found, forexample, in tissue engineering, in order to repair soft tissue,cartilage or bone defects. Another potential application is to fillcerebral aneurysms, taking advantage of the hemostatic capability ofchitosan. Additionally, injectable N-acylchitosan gels may be used tofill irregularly-shaped tissue defects or to improve the facial textureby treating wrinkles, creases, furrows, sunken cheeks, or scars. Anadvantage of injectable gels such as N-acylchitosan gels is that theymay be used to repair tissue defects in a minimally-invasive manner.These gels may also be used to deliver therapeutic agents, such as drugsto treat cancer, infections, inflammations, pain, and diseases/disordersof the central nervous system.

As described, extrusion may be performed using solutions or gels. Moregenerally, different types of mixtures may be extruded. This alsoincludes well-homogenized mixtures comprising different phases orsuspensions. The extrudate may contain additives including, for example,acids, bases, plasticizers, fillers, dyes, porogens, contrast agents,microparticles, nanoparticles, bioactive agents and drugs. Suchadditives may be added to the reaction mixture prior to gel formation,and/or may be added to the acylation bath in which the gel is formedupon extrusion.

In accordance with the present invention, the extrusion process mayinvolve a subsequent hydrolysis step of the extruded product, therebyforming a product having improved mechanical strength. Generally,hydrolysis may be performed before or after drying of the extrudedproduct. Hydrolysis (deacylation) may lead to a product having a verylow degree of acylation which increases the mechanical strength.Hydrolysis may be performed by storage of the extruded N-acylchitosangel in a concentrate alkaline solution at elevated temperature, such asfor example in 40% aqueous sodium hydroxide solution at 110.degree. C.for 2 hours (Example 3, FIG. 3). More generally, hydrolysis may beperformed by storage of N-acylchitosan in a 10-50% aqueous alkalinesolution at 50-120.degree. C. for up to 4 hours. Preferably, hydrolysismay be performed using a 20-40% aqueous alkaline solution at70-110.degree. C. for 0.5-2 hours. Hydrolysis may also be performed inseveral cycles in order to further decrease the degree of acylation andimprove the mechanical strength under physiological conditions.Preferably, 1-3 cycles of hydrolysis may be used according to thepresent invention (Example 3, FIG. 4). Additionally, hydrolysis may beparticularly suitable for extruded N-acylchitosan gels formed by usingacyl anhydrides different from acetic anhydride, in order to completelyremove the acyl group and re-establishing the natural chemical structureof the biopolymer.

It is in the scope of the present invention to include also thoseN-acylchitosan gels which are essentially free of N-acyl groups due toseveral cycles of hydrolysis. It will be understood by those skilled inthe art that 100% deacylation of N-acylchitosan is only of theoreticalinterest and that in practice even in materials which are essentiallyfree of N- acyl groups some N-acyl groups still remain in the chemicalstructure so that these materials are also referred to as N-acylchitosanwith respect to the present invention.

In accordance with the present invention, the extrusion process may alsoinvolve a subsequent drying step of the extruded product, therebyforming a product having improved mechanical strength (Example 4, FIG.5). N-acylchitosan hydrogels are highly porous, comprising ahoneycomb-like morphology which collapses irreversibly during drying,leading to a denser packing of the polymer bulk and higher mechanicalstrength under physiological conditions. It should be understood that“drying” with respect to the present invention includes processesleading to the removal of liquid components including water and othersolvents from the hydrogel. Drying may preferably be performed bystorage of the extruded N-acylchitosan gel product on air and at roomtemperature. Similarly, any gas which may flow or not, may be used forthe drying process. Other methods include the application of highertemperatures and/or vacuum during storage. The temperatures and methodschosen for drying should be appropriate to prevent decomposition,cross-linking and other changes in the chemical structure of theN-acylchitosan gel. Drying may be performed completely, which may takeseveral minutes to hours depending on the geometry and thickness of theproduct, or partially, for a defined period of time, in order to definethe properties of the final product. After fabrication of driedarticles, they may simply be stored in a dry atmosphere, or they may bestored in an aqueous environment. Dried articles for medicalapplications can easily be sterilized, preferably with ethylene oxide,hydrogen peroxide plasma, or gamma irradiation. Dried articles areusually relatively stiff, but become flexible when re-immersed inaqueous solution which facilitates their handling, includingimplantation into a body, and which allows for comfort to the patientduring and after implantation.

In accordance with the present invention, the extruded product may alsobe modified by coating with a layer of a polymer or other compound,which may be applied from solution by one of the techniques well-knownin the art, such as dipping or spraying. Generally, coating may beapplied before or after drying of the extruded product. Additionally,coating may be applied to the hydrolyzed product, before or after itsdrying. For example, a layer of a biodegradable polymer may be formed onthe surface of a product in order to control its properties, includingmechanical strength, biocompatibility, biodegradation, diffusibility,and adsorption properties. Suitable biodegradable polymers include, forexample, those from the group of synthetic polyesters, such ashomopolymers and copolymers based on glycolide, L-lactide, D,L-lactide,p-dioxanone, E-caprolactone; natural polyesters, such as those from thegroup of the polyhydroxyalkanoates, such as homopolymers and copolymersbased on 3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxyvalerate,3-hydroxyhexanoate, 3-hydroxyoctanoate; polyorthoesters; polycarbonates;polyanhydrides; polyurethanes; polyphosphazenes; polyphosphoesters;polysaccharides; polypeptides; as well as derivatives, copolymers, andblends based on the abovementioned and any other group of bioresorbablepolymers. Other suitable polymers include those which may be dissolvedunder physiological conditions, such as homopolymers or copolymers basedon vinyl alcohol, vinyl acetate, N-vinyl pyrrolidone, ethylene glycol,propylene glycol, polysaccharides, polypeptides, as well as derivatives,copolymers, and blends based on the aforementioned and any other groupof biodissolvable polymers or combinations of biodegradable andbiodissolvable polymers. Furthermore, it is possible to coat the articlewith a non-degradable or non-dissolvable polymer for specificapplications of the extruded article, which require to prevent itsdegradation.

The polymer layer may further contain additives, including acids, bases,plasticizers, fillers, dyes, porogens, contrast agents, microparticles,nanoparticles, bioactive agents and drugs. Such additives may be addedto the polymer solution prior to the coating process. In anotherexample, a layer of a contrast agent may be formed on the surface of theextruded article after its fabrication. For example, a layer of bariumsulfate may be formed by dipping the article into an aqueous solution ofa barium salt, followed by dipping into an aqueous solution containingsulfate ions, thereby forming a layer of barium sulfate on the surfaceof the article. In yet another example, a layer of a bioactive agent ordrug may be formed on the surface of the extruded article. For example,a layer of a bioactive agent or drug may be applied to the surface usingan aqueous solution or organic solvent, followed by drying.

In accordance with the present invention, there is provided an extrusionprocess for the fabrication of N-acylchitosan gels, fibers, tubes,films, and other articles, which may be hollow or not. Tubes may have asingle lumen or multiple lumens, and they may be single- ormultilayered. Tube extrusion may involve a gaseous, liquid or solid corecomponent for stabilizing the internal tube dimensions, as it is knownin the art. The initial shape of the extruded product is defined by thedesign of the die used in the extrusion process. Die designs arewell-known in the art and include sheet dies, profile dies, tubing dies,and coating dies. The variation of die designs allows for the extrusionof N-acylchitosan gel articles in different cross-sectional shapes, suchas the fabrication of cylindrical, triangular, quadrangular, and ingeneral, polygonal tubes. Moreover, as it has been outlined above andalso shown on the example of N-acetylchitosan gel tubes (FIG. 5), dryingof the extruded product results in improved mechanical strength. Thehigher compression strength of tubes after drying (see FIG. 5) furtherimplies a higher stability of the tubular (cross-sectional) shape. Suchfor example, a dried tube which is fabricated according to the presentinvention can be cut open longitudinally without losing its tubular(cross-sectional) shape stability which would be, for example, ofadvantage in applications as a wrap around structures to be stabilized,such as damaged or diseased nerve.

However, extrusion through a die does not provide any means to formspecific designs (shapes) in the longitudinal direction of the extrudedN-acylchitosan gel article. It is therefore another preferred embodimentof the present invention to provide shaped articles based onN-acylchitosan having improved mechanical strength and shape-memorystability not only in the cross-sectional but also longitudinaldirection. Furthermore, it is in the scope of the present invention toprovide a process for implementing shape-memorized designs intoN-acylchitosan articles without affecting biocompatibility andbiodegradation properties which is of particular importance for medicaldevices. Shaped medical devices according to this embodiment of thepresent invention may include those based on fibers, including coils,dressings, meshes, gauzes and similar structures. Furthermore, shapedmedical devices according to this embodiment of the present inventionmay include those based on tubular structures, including biliary,urinary and vascular stents, catheters, cannulas and similar devices.Additionally, they may include screws, plates, rods, anchors, plugs,fillers, capsules and any other shaped medical device. Preferred devicesinclude sutures, suture fasteners, slings, coils, rivets, tacks,staples, clips, hooks, buttons, snaps, orthopedicpins/clamps/screws/dowels/plates, bone substitutes, spinalcages/plates/rods/screws/discs, finger joints, intramedullary nails, hipprosthesis, meniscus repair devices, knee replacement devices, cartilagerepair devices, ligament and tendon grafts, tendon repair devices,surgical mesh, surgical repair patches, hernia patches, pericardialpatches, cardiovascular patches, adhesion barriers, abdominal wallprosthesis, catheters, shunts, stents (coronary, gastrointestinal,esophageal, biliary, ureteral, urethral, stents for aortic aneurysms),vascular grafts and substitutes, coronary artery bypass grafts, guidedtissue repair/regeneration devices, scaffolds for tissue engineering,nerve guides, septal defect repair devices, heart valves, vein valves,artificial fallopian tubes, drainage tubes and implants, intrauterinedevices, intraocular implants, keratoprosthesis, dental implants,orbital floor substitutes, skin substitutes, dural substitutes,intestinal substitutes, fascial substitutes, wound dressings, burndressings, medicated dressings, gauze/fabric/sheet/felt/sponge forhemostasis, gauze bandages, bandages for skin surfaces, adhesivebandages, bulking and filling agents, drug delivery matrices, injectablegels and systems, coatings applied to pacemaker leads, implantablesensor wire leads, wires for interventional cardiology, or biosensors,and others.

The shaped medical devices according to this embodiment of the presentinvention are particularly applicable for use in urogenital,cardiovascular, gastrointestinal, neurological, lymphatic,otorhinolaryngological, opthalmological and dental applications.Additionally, they are particularly interesting for applications intissue engineering, including those containing steps of cellularseeding.

The shaped articles according to this embodiment of the presentinvention are made by starting from N-acylchitosan gels. Generally,N-acylchitosan gels used to fabricate shaped articles are made byextrusion in accordance with the present invention. Additionally,N-acylchitosan gels made by other processes which are known in the artmay be suitable to fabricate shaped articles. Injection molding is themost preferable method among these other processes.

Preferred gels for the fabrication of shaped articles according to thisembodiment of the present invention are those consisting ofN-acetylchitosan. Preferably, extrusion involves dissolution of 2-10%chitosan in 0.5-15% aqueous acetic acid, addition of a 1-2.5 fold volumeof ethanol, and extrusion of the resulting homogeneous mixture into anacylation bath containing 10-90% acetic anhydride in ethanol, asdescribed above and illustrated in Example 1. More preferably, chitosanis dissolved in a concentration of 3-5% in 2-5% aqueous acetic acid,mixed with a 1-2 fold volume of ethanol, and extruded into an acylationbath containing 25-50% of acetic anhydride in ethanol. Methanol may bebeneficial over ethanol under specific conditions, particularly ifhigher concentrations of chitosan are used. In this case, chitosan ispreferably dissolved in a concentration of 5-7% in 2-5% aqueous aceticacid, mixed with a 1-2 fold volume of methanol, and extruded into anacylation bath containing 25-50% of acetic anhydride in either ethanolor methanol. For injection-molding, N-acetylchitosan gels are preferablymade by treatment of a solution of 2-5% chitosan in 0.5-10% aqueousacetic acid, the solution being diluted with a 0.5-2 fold volume ofethanol, with a 1-3 fold excess of acetic anhydride. More preferably, asolution of 3-4% chitosan in 2-5% aqueous acetic acid is mixed with a1-2 fold volume of ethanol, and a 1.5-2.5 fold excess of aceticanhydride is added, as illustrated in Example 5.

In both cases, for extrusion and injection-molding, the chitosan used asstarting material has preferably a degree of acetylation of less than25% and a viscosity between approximately 200-2000 mPas (analyzed as 1%solution in 1% acetic acid on a Brookfield viscometer at 25.degree. C.).More preferably, the chitosan has a degree of acetylation of less than15% and a viscosity between approximately 200-1000 mPas.

N-acylchitosan gels which are suitable for the fabrication of shapedarticles according to this embodiment of the present invention may havethe shape of a rod, fiber, tube, film, sphere or other geometricstructures which may be hollow or not. The gel may already have a shapesimilar to that of the desired final product. Fibers, tubes, films, andother articles, which may be hollow or not, may be made by extrusion, asdescribed above and illustrated in Example 1. In an injection-moldingprocess, as illustrated in Example 3, the acylation reaction mixture maysimply be injected into a mold of pre-selected size and shape, and willbe left for gelation without further application of any forces, in orderto allow for homogeneous gel formation. For example, movement of themold or application of forces to the mold during gel formation mayresult in inhomogenic gel morphologies which is disadvantageous withrespect to the formation of shaped articles according to the presentinvention. N-acylchitosan gel rods and fibers may be fabricated byinjecting the acylation reaction mixture into a cylindrical mold for gelformation (Example 5). Similarly, N-acylchitosan gel tubes may befabricated by injecting the acylation reaction mixture into acylindrical mold which contains a centrally fixed core for gel formation(Example 5). Cylindrical molds may contain more than one core tofabricate gel tubes with multiple channels. Corrugated rods and tubesmay be fabricated by using a corrugated mold for injection and gelformation. Similarly, other three-dimensional structures may befabricated by injecting the acylation reaction mixture into appropriatemolds for gelation. N-acylchitosan gel films can simply be made bypouring the acylation reaction mixture into a Petri dish or similarcontainer for gel formation, or by injection into a suitable mold.Another technique is to cut a gel tube longitudinally to provide a filmfor further processing into a shaped article according to the presentinvention.

Shaped articles according to this embodiment of the present inventionhaving improved mechanical strength and shape-memory stability arefabricated by drying N-acylchitosan gel structures such as thosedescribed above under fixation of the desired shape. The collapse of thehoneycomb-like morphology of the hydrogel during thedehydration/desolvation process leads to the irreversible preservationof the fixed shape together with improved mechanical stability due to adenser packing of the polymer bulk.

It should be understood that “drying” with respect to the fabrication ofshaped articles according to this embodiment of the present inventionincludes processes leading to the removal of liquid components includingwater and other solvents from the hydrogel. Drying may preferably beperformed by storage of the shape-fixed N-acylchitosan gel on air and atroom temperature. Similarly, any gas which may flow or not, may be usedfor the drying process. Other methods include the application of highertemperatures and/or vacuum during storage. The temperatures and methodschosen for drying should be appropriate to prevent decomposition,cross-linking and other changes in the chemical structure of theN-acylchitosan gel. Drying may be performed completely, which may takeseveral minutes to hours depending on the geometry and thickness of theshape-fixed article, or partially, for a defined period of time, inorder to define the properties of the final product. It is important tonote that freeze-drying or other processes of drying which prevent thecollapse of the honeycomb-like morphology of the gel do not allow forfabrication of articles with suitable mechanical strength andshape-memory properties according to the present invention. Afterfabrication of dried articles, they may simply be stored in a dryatmosphere, or they may be stored in an aqueous environment. Driedarticles can easily be sterilized, preferably with ethylene oxide,hydrogen peroxide plasma, or gamma irradiation. Dried articles areusually relatively stiff, but become flexible when re-immersed inaqueous solution which facilitates their handling, includingimplantation into a body, and which allows for comfort to the patientduring and after implantation.

N-acylchitosan gels suitable for the fabrication of shaped articlesaccording to this embodiment of the present invention may be modifiedprior to the drying process. The modification may include ionic orcovalent binding of a compound, such as a bioactive agent or drug. Othermodifications include controlled acylation or hydrolysis reactions, inorder to adjust the degree of acylation of the gel, thereby controllingmechanical properties, biodegradation, and biocompatibility. Mostpreferable is a hydrolysis (deacylation) reaction leading to productshaving a low degree of acylation which further increases the mechanicalstrength. Hydrolysis may be performed by storage of the gel inconcentrated alkaline solutions at elevated temperatures, such as forexample in 40% aqueous sodium hydroxide solution at 110.degree. C. for 2hours. More generally, hydrolysis may be performed by storage ofN-acylchitosan in a 10-50% aqueous alkaline solution at 50-120.degree.C. for up to 4 hours. Preferably, hydrolysis may be performed using a20-40% aqueous alkaline solution at 70-110.degree. C. for 0.5-2 hours.Hydrolysis may also be performed in several cycles in order to furtherdecrease the degree of acylation and improve the mechanical strength.Preferably, 1-3 cycles of hydrolysis may be used according to thepresent invention. Additionally, hydrolysis may be particularly suitablefor gels formed by using acyl anhydrides different from aceticanhydride, in order to completely remove the acyl group andre-establishing the natural chemical structure of the biopolymer.

The shaped article in accordance with this embodiment of the presentinvention may contain additives, allowing the article to be designed tothe specific requirements. Such additives may include acids, bases,plasticizers, fillers, dyes, porogens, contrast agents, microparticles,nanoparticles, bioactive agents and drugs. Such additives may be addedto the reaction mixture prior to gel formation, and/or may be soakedinto the gel by storage of the gel in a solution of the additive priorto the drying process. Such additives may also be soaked into the bulkor coated onto the surface of the product after drying.

The shaped article fabricated in accordance with this embodiment of thepresent invention may further be modified by a method described abovefor N-acylchitosan gels, including ionic or covalent binding of acompound, such as a bioactive agent or drug, and controlled acylation orhydrolysis reactions, in order to adjust the degree of acylation of thearticle, thereby controlling mechanical properties, biodegradation, andbiocompatibility. Most preferable is a hydrolysis (deacylation) reactionleading to products having a very low degree of acylation which furtherincreases the mechanical strength. Hydrolysis may be performed bystorage of the article in concentrated alkaline solutions at elevatedtemperatures, such as for example in 40% aqueous sodium hydroxidesolution at 110.degree. C. for 2 hours. More generally, hydrolysis maybe performed by storage of N-acylchitosan in a 10-50% aqueous alkalinesolution at 50-120.degree. C. for up to 4 hours. Preferably, hydrolysismay be performed using a 20-40% aqueous alkaline solution at70-110.degree. C. for 0.5-2 hours. Hydrolysis may also be performed inseveral cycles in order to further decrease the degree of acylation.Preferably, 1-3 cycles of hydrolysis may be used according to thepresent invention. Additionally, hydrolysis may be particularly suitablefor articles formed by using acyl anhydrides different from aceticanhydride, in order to remove the acyl group and re-establishing thenatural chemical structure of the biopolymer.

The shaped article may also be modified by coating with a layer of apolymer or other compound, which may be applied from solution by one ofthe techniques well-known in the art, such as dipping or spraying. Thusfor example, a layer of a biodegradable polymer may be formed on thesurface of the shaped article in order to control its properties,including mechanical strength, biocompatibility, and biodegradation.Suitable biodegradable polymers include, for example, those from thegroup of synthetic polyesters, such as homopolymers and copolymers basedon glycolide, L-lactide, D,L-lactide, p-dioxanone, E-caprolactone;natural polyesters, such as those from the group of thepolyhydroxyalkanoates, such as homopolymers and copolymers based on3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxyvalerate,3-hydroxyhexanoate, 3 hydroxyoctanoate; polyorthoesters; polycarbonates;polyanhydrides; polyurethanes; polyphosphazenes; polyphosphoesters;polysaccharides; polypeptides; as well as derivatives, copolymers, andblends based on the abovementioned and any other group of bioresorbablepolymers. Other suitable polymers include those which may be dissolvedunder physiological conditions, such as homopolymers or copolymers basedon vinyl alcohol, vinyl acetate, N-vinyl pyrrolidone, ethylene glycol,propylene glycol, polysaccharides, polypeptides, as well as derivatives,copolymers, and blends based on the aforementioned and any other groupof biodissolvable polymers or combinations of biodegradable andbiodissolvable polymers. Furthermore, it is possible to coat the shapedarticle with a non-degradable or non-dissolvable polymer for specificapplications of the shaped articles, which require to prevent itsdegradation.

The polymer layer may further contain additives, including acids, bases,plasticizers, fillers, dyes, porogens, contrast agents, microparticles,nanoparticles, bioactive agents and drugs. Such additives may be addedto the polymer solution prior to the coating process. In anotherexample, a layer of a contrast agent may be formed on the surface of theshaped article after its fabrication. For example, a layer of bariumsulfate may be formed by dipping the shaped article into an aqueoussolution of a barium salt, followed by dipping into an aqueous solutioncontaining sulfate ions, thereby forming a layer of barium sulfate onthe surface of the shaped article. In yet another example, a layer of abioactive agent or drug may be formed on the surface of the shapedarticle. For example, a layer of a bioactive agent or drug may beapplied to the surface using an aqueous solution or organic solvent,followed by drying.

In yet another example of modification of the shaped article, anadditional layer of N-acylchitosan gel may be applied to the surface ofthe article and may be dried for shape-fixation. These steps may berepeated several times to fabricate a multilayered article. TheN-acylchitosan layers may have different properties such as differentdegrees of acylation in order to define individual mechanical,biocompatibility, and biodegradation properties of individual layers.The N-acylchitosan layers may be modified by techniques as describedabove or may contain additives as those described above. Such additivesmay also be embedded between the layers. In such a design, the additivewill be applied to the surface of one layer of the article before addingthe next layer of N-acylchitosan gel. The subsequent drying process ofthis outer layer will lead to the incorporation of the additive betweenthe layers.

In accordance with this embodiment of the present invention, the shapedarticle is conformable to the shape of a medical device or part of amedical device, including the shape of an anchor, hook, coil, mesh,textile, foam, scaffold, stent, catheter, tube, sphere, particle, plug,plate, screw, pin, tack, clip, ring, drug-release depot,cell-encapsulation device.

For example, a coil may be fabricated by winding an N-acylchitosan gelfiber of defined diameter on a mandrel or screw, fixing the ends of thefiber to maintain the coiled shape, and drying by storage on air,leading to a shape-memorized coiled conformation of the resultingarticle (Example 6, FIG. 6). More complex three-dimensional structuresmay be fabricated by drying of N-acylchitosan gel fibers fixed indefined conformations using complex designs of mandrels such as thoseconsisting of a center post with side pins as known in the art. A hollowcoil or spiral may be fabricated by winding and fixing an N-acylchitosangel tube on a mandrel for drying (Example 7, FIG. 7). Tubular articlessuch as stents and catheters may be fabricated by fixing anN-acylchitosan gel tube in the desired shape during drying. Thusshape-memorized tubular designs may have one or more sections which areshape-memorized in a non-linear conformation with respect to thelongitudinal axis of the tube. One particularly interesting shape of atubular article is that of a ureteral stent having ends in the form of apigtail (“double-J catheter”) (Example 8, FIG. 8). This type of stentmay be fabricated by fixing the ends of an N-acylchitosan gel tube inthe desired pigtail conformation during the drying process, leading to ashape-memorized pigtail conformation of the resulting article. Anothershaped article of particular interest is a Foley catheter, which may beformed by a combination of a two-channel tube and a hollow sphere madeof N-acylchitosan (Example 9, FIGS. 9A and 9B). It should be noted thatother distorted hollow structures may be used for fabrication of thistype of catheter, including ovate, ovoid, or ellipsoid shapes. It isworth noting that urological articles such as stents and catheters wouldbe highly beneficial when made of N-acetylchitosan compared to othermaterials, due to the inherent antibacterial properties ofN-acetylchitosan, thereby limiting and preventing infections which arecommonly associated with these implants. N-acetylchitosan may also behighly beneficial as a coating of other materials used in urologicalapplications.

In accordance with this embodiment of the present invention, the shapedarticle may be formed as a coating, made by starting from anN-acylchitosan gel, of a medical device or part of a medical device,including an anchor, hook, fiber (including fiber bundles), coil, mesh,textile, foam, sponge, scaffold, stent, catheter, tube, sphere,particle, plug, plate, screw, pin, tack, clip, ring, drug-release depot,cell-encapsulation device. This coating may be performed by attaching anN-acylchitosan gel, which may have the shape of a rod, fiber, coil,tube, film, sphere or other geometric structures, to the surface of thedevice or part of a device to be coated, followed by drying. One or moredevices may be coated together in one step.

Such for example, a coil may be coated by inserting it into anN-acylchitosan gel tube which has an inner diameter slightly larger thanthe outer diameter of the coil, and drying (Example 10, FIG. 10). Rods,including cylindrical drug pellets (Example 11, FIG. 11), and tubes,including porous tubes (Example 12, FIG. 12) may be coated in a similarmanner. As an example, a stent or catheter may be inserted into anN-acylchitosan gel tube of appropriate dimensions, i.e. with an innerdiameter slightly larger than the outer diameter of the stent orcatheter. The tube will then be dried for shape-fixation and attachmentto the underlying stent or catheter. A screw or pin may be insertedsimilarly into an N-acylchitosan gel tube, followed by its dryingleading to attachment to the screw or pin (Example 13, FIG. 13). Fibersand fiber bundles, porous structures, foams, sponges, powders, pellets,or tablets may be coated in a similar manner, by inserting in anN-acylchitosan gel tube or other hollow structure of appropriatedimension, followed by drying. As another example, a fiber or fiberbundle may be inserted into an N-acylchitosan gel tube, and thefiber/gel tube composite will be fixed in a desired shape, such as acoiled shape as described above, for drying and shape-fixation.

Coating may also be performed by dipping a medical device or part of amedical device, including those mentioned above, into the acylationreaction medium, followed by removal and gel layer formation on thesurface of the dipped material. Subsequent drying leads toshape-fixation of the N-acylchitosan coating. Modifications as thosedescribed above may be applied before and/or after drying, as describedabove.

EXAMPLES 1. Extrusion of N-acetylchitosan Gel Fibers and Tubes

A 3% solution of chitosan (degree of acetylation 11%) in 2% aqueousacetic acid was diluted with an equal volume of ethanol. A 0.5 foldmolar amount of acetic anhydride was added to the solution. The reactionmixture was filtered and degassed, and extruded through a single-holespinneret (inner diameter 0.25 mm) into a bath containing 50% aceticanhydride in ethanol. The N-acetylchitosan gel fiber formed was washedwith distilled water, and air-dried resulting in an N-acetylchitosanfiber having a diameter of approximately 50 .mu.m. N-Acetylchitosanhollow fibers (tubes) were fabricated in a similar manner, by extrudingthe reaction mixture through a spinneret comprising an annular ring(outer diameter 2.3 mm, width of the annular gap 0.25 mm) through whichthe chitosan solution passed, and a central bore (diameter 1.5 mm)through which a core liquid consisting of 50% acetic anhydride inethanol was delivered. The N-acetylchitosan gel tube formed was washedwith distilled water, and stored in a closed container containingsterile distilled water.

In another experiment, N-acetylchitosan fibers were made by startingfrom a solution of 6% chitosan (degree of acetylation 11%) in 2% aqueousacetic acid, which was diluted with an equal volume of methanol. Thereaction mixture was filtered and degassed, and extruded through asingle-hole spinneret (inner diameter 0.25 mm) into a bath containing50% acetic anhydride in methanol. The N-acetylchitosan gel fiber formedwas washed with distilled water, and air-dried resulting in anN-acetylchitosan fiber having a diameter of approximately 100 .mu.m.

2. Fabrication of Injectable Gels

A 3% solution of chitosan (degree of acetylation 11%) in 2% aqueousacetic acid was diluted with a twofold volume of ethanol. A 0.5 foldmolar amount of acetic anhydride was added to the solution. The reactionmixture was sonicated to remove air-bubbles and transferred into asyringe. The resulting gel was extruded through a 30 gauge needleforming a gel-like extrudate.

3. Hydrolysis of N-acetylchitosan Gel Tubes

N-Acetylchitosan gel tubes were mounted on a cylindrical glass core(outer diameter 1.5 mm) and stored in a 40% aqueous solution of sodiumhydroxide at 110.degree. C. for 2 h. The tubes were then intenselywashed with distilled water, and air-dried for 3 h at room temperatureand normal pressure. The degree of acetylation, as tested by NMR,decreased from 94% to 18% during hydrolysis (FIG. 3). To achieve greaterlevels of deacetylation, tubes were stored for 2 h in fresh alkalinesolution as described above, followed by washing with water. This cycleof hydrolysis/washing was repeated up to 2 times. Degrees of acetylationof 3% and 1% were achieved after the second and third hydrolysis cycle.Hydrolysis resulted in tubes of high mechanical strength underphysiological conditions (FIG. 4).

4. Drying of N-acetylchitosan Gel Tubes

Dried N-acetylchitosan tubes were fabricated by storing N-acetylchitosangel tubes on air for 3 h at room temperature and normal pressure. Acylindrical glass core (diameter 1.5 mm), which was pre-coated with athin layer of poly(.epsilon.-caprolactone) in order to facilitatesubsequent tube removal, was inserted into the tubes during drying.Mechanical testing of tubes showed increased compression strength ofdried tubes compared to hydrogels, despite smaller wall thicknesses,under physiological conditions (FIG. 5). Sterilization with ethyleneoxide resulted in no significant changes in the compression strength.

In alternative experiments, small-diameter N-acetylchitosan tubes werefabricated by drying N-acetylchitosan gel tubes after mounting onplatinum wire (diameter 125 .mu.m) under conditions as described above,resulting in tubes with an inner diameter of 125 .mu.m. Similarly,platinum wire of 25 .mu.m has been used to fabricate tubes with an innerdiameter of 25 .mu.m. Alternatively, stainless steel and copper havebeen used for tube fabrication.

5. Injection-molding of N-acetylchitosan Gel Fibers and Tubes

A 3% solution of chitosan (degree of acetylation 11%) in 2% aqueousacetic acid was diluted with an equal volume of ethanol and, aftercooling to about 10.degree. C., mixed with a twofold molar excess ofacetic anhydride. The reaction mixture was sonicated to removeair-bubbles and injected into a sealed cylindrical glass mold (innerdiameter 0.8 mm). Gelation occurred within approximately 3 min and after24 h, during which syneresis occurred, the N-acetylchitosan hydrogelfiber was removed from the mold and first washed with, and then storedin, water. Tubes were prepared in a similar manner, by using acylindrical glass mold (inner diameter 4.0 mm), which contained a fixedcentral cylindrical glass core (diameter 1.7 mm).

6. Fabrication of Fiber Coils (FIG. 6)

N-Acetylchitosan gel fibers, fabricated as described in Example 1, werewound on a cylindrical mandrel, and the ends were fixed. After dryingfor 3 h at room temperature and normal pressure, the resultingshape-memorized N-acetylchitosan coil was re-immersed in water, removedfrom the mandrel, re-dried and stored in a closed container.

7. Fabrication of Hollow Fiber Coils (FIG. 7)

N-Acetylchitosan gel tubes were fabricated as described in Example 1. Acylindrical silicon core was inserted into the tube lumen. The tube/coreassembling was wound on a cylindrical mandrel, and the ends were fixed.After drying for 3 h at room temperature and normal pressure, theresulting shape-memorized N-acetylchitosan coil was re-immersed inwater, removed from the mandrel and the silicon core, and stored indistilled water. Similar results were obtained using platinum wireinstead of silicon for insertion into the gel tube prior to winding anddrying.

8. Fabrication of Ureteral Stents (FIG. 8)

N-Acetylchitosan gel tubes were fabricated as described in Example 1. Acylindrical silicon core was inserted into the tube lumen, and the endsof the tube/core assembling were fixed in a pigtail conformation. Afterdrying for 3 h at room temperature and normal pressure, the resultingN-acetylchitosan ureteral stent having shape-memorized pigtail ends wasre-immersed in water, removed from the silicon core, re-dried and storedin a closed container. Similar results were obtained using platinum wireinstead of silicon for insertion into the gel tube prior to pigtailshape-fixation and drying. N-Acetylchitosan ureteral stents were furthersubjected to hydrolysis reactions. For this, the tube/core assemblingfixed in a pigtail conformation and dried as described above wasimmersed in a 40% aqueous solution of sodium hydroxide at 110.degree. C.for 2 h. This step was repeated up to two times. The stent was washedintensely in distilled water, the silicon core removed, the stent driedand stored in a closed container. Similarly, platinum wire could be usedfor insertion into the N-acetylchitosan tube lumen during hydrolysisreactions.

Alternative experiments included hydrolysis of pigtail shape-fixedN-acetylchitosan gel tubes under conditions described above for driedtubes. In another alternative experiment, N-acetylchitosan gel tubeswere hydrolyzed without any shape-fixation as described above. The tubeends were then fixed in pigtail conformation and dried similarly to theprocedures described above.

In another experiment, N-acetylchitosan ureteral stents were coated witha polymer layer in order to adjust degradation times. For example,stents were dip-coated with a solution of poly(D,L-lactide-co-glycolide)in acetone. Preferable concentrations of the polymer solution werebetween 0.5% and 2%.

In yet another experiment, N-acetylchitosan ureteral stents were coatedwith a layer of barium sulfate, by dipping the stents into an aqueoussuspension of barium sulfate, removal, and drying. Additionally, stentscould be perforated, as illustrated in FIG. 8.

N-acetylchitosan ureteral stents have been tested for theirbiodegradation properties in vitro. In these experiments, stents werestored in human urine, which was replaced daily, at 37.degree. C.Results from these experiments are summarized in Table 1. Non-hydrolyzedstents showed stiffness and increasing brittleness includingfragmentation into brittle pieces which would lead to discomfort in apatient. Stents hydrolyzed for 2 h in 40% aqueous sodium hydroxide at110.degree. C. and coated with 0.5% PLGA showed a favourable degradationprofile with respect to the target application: on day 3 the tubeswelled and became soft; increasing softness was observed between days 3and 9; beginning fragmentation into gel-like pieces at day 9; completedegradation at day 12. Such a degradation profile is highly desirable toallow for maximum functional efficacy and patient comfort duringimplantation and avoid a second surgical treatment. An advantage ofstents hydrolyzed three times is their pH-dependent dissolutionmechanism. These stents can be removed from the patients body in ahighly controllable fashion, by adjusting the pH of the patient's urine,which can be done by treatment with basic or acidic compounds added tothe diet. These methods of pH-adjustment of the urine are well-known inthe clinical practice. For example, a basic pH may be maintained bygiving a base or basic salt, such as acetazolamide or bicarbonate, tothe patient for a desired period of time, e.g. 2 weeks, after which theurine may be made acidic (if not naturally back-regulated to an acidicpH) by giving an acid or acidic salt, such as ammonium chloride, whichwill, at a pH of less than about 6, result in a fast dissolution of thestent and its disappearance from the body. A general feature of allN-acetylchitosan modifications tested with respect to this invention istheir inherent antibacterial potential which makes this class ofmaterials highly promising for urological applications to prevent stent-or catheter-related infections.

TABLE-US-00001 TABLE 1 In vitro degradation/dissolution profile ofN-acetylchitosan ureteral stents (urine, 37.degree. C.).N-Acetylchitosan Mechanical Properties ureteral stent Degradation Timeduring Degradation Non-hydrolyzed, 2 weeks increasing stiffness andnon-coated brittleness Non-hydrolyzed, >10 weeks increasing stiffnessand PLGA coated brittleness 1 .times. 2 h Hydrolysis, 3-5 daysincreasing softness and non-coated gel-like dissolution 1 .times. 2 hHydrolysis, 10-12 days increasing softness and PLGA coated gel-likedissolution 3 .times. 2 h Hydrolysis, pH dependent stiffness essentiallynon-coated (2 days in acidic urine) unchanged, not brittle 3 .times. 2 hHydrolysis, pH dependent stiffness essentially PLGA coated unchanged,not brittle

9. Fabrication of Foley Catheters (FIGS. 9A and 9B)

N-Acetylchitosan gel tubes were fabricated in a modified procedure tothat described in Example 5, by injecting the acetylation reactionmixture into a cylindrical mold (inner diameter 5.5 mm), which containedtwo intraluminally fixed cylindrical cores (outer diameter 3.3 mm and0.6 mm, respectively). Gelation and air-drying resulted in a two-channelN-acetylchitosan tube. The tubes were perforated, as illustrated in FIG.9A. N-Acetylchitosan gel hollow spheres were fabricated by dipping aninflated polymeric balloon into the acetylation reaction mixture,removal from the mixture for gel formation on the outside of theinflated balloon, and air drying. The balloon was then deflated andremoved from the formed hollow sphere, which was then mounted on thetube and fixed using a glue.

10. Coating of Coil-shaped Medical Devices (FIG. 10)

N-Acetylchitosan gel tubes were fabricated as described in Example 1. Ahelical coil was mounted on a cylindrical core, and the coil/coreassembling was inserted into the tube. Air-drying of the gel resulted inthe formation of a tubular N-acetylchitosan coating of the coil.

11. Coating of Drug-Delivery Depots (FIG. 11)

N-Acetylchitosan gel tubes were fabricated as described in Example 1. Acylindrical drug pellet was inserted into the tube. Air-drying of thegel resulted in the formation of a tubular N-acetylchitosan coating ofthe pellet.

12. Coating of Medical Catheters (FIG. 12)

N-Acetylchitosan gel tubes were fabricated as described in Example 1. Acatheter was inserted into the tube. Air-drying of the gel resulted inthe formation of a tubular N-acetylchitosan coating of the catheter.

An alternative method, dipping of the catheter into the acetylationreaction mixture, removal for gel formation and air-drying of the gelformed on the surface of the catheter, has also been tested. The ends ofthe catheter have been closed with plugs prior to dipping to preventintraluminal coating.

13. Coating of Medical Screws (FIG. 13)

N-Acetylchitosan gel tubes were fabricated as described in Example 1. Ascrew was inserted into the tube. Air-drying resulted in the formationof a corrugated N-acetylchitosan coating of the screw.

What is claimed is:
 1. A process for manufacturing an article comprisingN-acylchitosan hydrogel, the process comprising the steps of: providinga mixture comprising chitosan and/or N-acylchitosan; and injecting themixture into a mold of pre-selected size and shape to form and shape theN-acylchitosan hydrogel.
 2. The process of claim 1, wherein the mold iscylindrical.
 3. The process of claim 2, wherein the mold comprises acentrally fixed core.
 4. A process for manufacturing an articlecomprising N-acylchitosan, the process comprising the steps of:providing an N-acylchitosan hydrogel fixed in a desired shape; and atleast partially drying the hydrogel, thereby forming an article with amemorized shape.
 5. The process of claim 4, wherein the step ofproviding an N-acylchitosan hydrogel fixed in a desired shape comprisesthe steps of providing a mixture comprising chitosan and/orN-acylchitosan, and extruding the mixture to form an N-acylchitosanhydrogel.
 6. The process of claim 5, wherein upon extrusion of themixture, the N-acylchitosan hydrogel is formed essentially instantly. 7.The process of claim 5, wherein the mixture comprises a drug.
 8. Theprocess of claim 5, wherein the step of extruding the mixture comprisesthe extrusion of the mixture into a hydrogelation medium capable ofpromoting the formation of an N-acylchitosan hydrogel.
 9. The process ofclaim 8, wherein the hydrogelation medium comprises an acylation agent.10. The process of claim 8, wherein the hydrogelation medium furthercomprises a drug.
 11. The process of claim 4, wherein the step ofproviding an N-acylchitosan hydrogel comprises the step of injecting orpouring a mixture comprising chitosan and/or N-acylchitosan into a mold.12. The process of claim 4, wherein the mixture comprises chitosan orN-acylchitosan dissolved in a diluted acid and a cosolvent.
 13. Theprocess of claim 4, wherein the step of providing an N-acylchitosanhydrogel comprises coating another article with an N-acylchitosanhydrogel.
 14. The process of claim 13, wherein the other article is amedical device.
 15. The process of claim 13, wherein the other articlecomprises a drug.
 16. The process of claim 4, wherein the mixturecomprising chitosan and/or N-acylchitosan further comprises an acylationagent.
 17. The process of claim 4, wherein the process furthercomprises, before the step of drying, at least one step of hydrolyzingto at least partially deacylate the N-acylchitosan.
 18. The process ofclaim 4, further comprising the step of coating the article with atleast one coating layer.
 19. The process of claim 18, wherein thecoating layer comprises a drug.
 20. A process for manufacturing atubular medical device, a stent, a catheter, a vascular graft, a nerveguide, a drainage tube, a fiber-based medical device, a suture, amedical device based on a mesh or a gauze, a wound dressing, or a tissueengineering scaffold, the process comprising the step of: forming anN-acylchitosan hydrogel.